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|Title: ||Functional complementation of sir2Δ yeast mutation by the human orthologous gene SIRT1|
|Authors: ||GAGLIO, DAVIDE|
|Tutor: ||Vittorioso, Paola|
|Issue Date: ||21-Jan-2014|
|Abstract: ||Sirtuins, class III histone deacetylases, are proteins homologous to the yeast protein Sir2p. Mammalian Sirt1 has
been shown to be involved in energy metabolism, brain functions, inflammation and aging through its deacetylase
activity, acting on both histone and non-histone substrates. In order to verify whether Sirt1 can replace Sir2p in the
yeast cells, we expressed the full-length human Sirt1 protein in S.cerevisiae sir2Δ mutant strain. The structure of
chromatin is basically maintained from yeast to human. Thus, yeast chromatin is a favourable environment to
evaluate, inhibit or activate an ectopic histone deacetylase activity in an in vivo substrate. Mutant sir2Δ shows a
series of different phenotypes, all dependent on the deacetylase activity of Sir2p. We analyzed the three silent loci
where normally Sir2p acts: ribosomal DNA, telomeres and the mating type loci. Moreover, we verified
extrachromosomal ribosomal DNA circles production and histone hyperacetylation levels, typical marks of sir2Δ
strains. By strong SIRT1 overexpression in sir2Δ cells, we found that specific molecular phenotypes of the mutant
revert almost to a wild-type condition. In particular, transcriptional silencing at rDNA was restored, extrachromosomal
rDNA circles formation was repressed and histone acetylation at H3K9 and H4K16 decreased. The complementation
at the other studied loci: HM loci, telomere and sub-telomere does not occur. Overall, our observations indicate that:
i) SIRT1 gene is able to complement different molecular phenotypes of the sir2Δ mutant at rDNA ii) the in vivo
screening of Sirt1 activity is possible in yeast.|
|Research interests: ||Molecular Biology, Cancer, Genome stability, Histone modifications, Chromatin remodeling, Transcriptional regulation, Ageing|
|Skills short description: ||Laboratory skills:
- Western Blot, Northern Blot, Southern Blot.
- ChIP, ChIP-seq, RNA-seq
- Gene and recombinant DNA cloning, recombinant protein for heterologous expression
(restriction enzymes, ligation, cell transformation, PCR screening etc..)
- PCR, semiquantitative sqRT-PCR, Colony PCR, real time qRT-PCR.
- DNA/RNA extraction, DNA/RNA precipitation, plasmid preparation Mini, Midi and Maxi
Prep, skilful in DNA/RNA/Protein handling of different organisms and cell lines.
- Agarose and polyacrylamide Gel Electrophoresis, DNA gel purification.
- Electroporation, Heat Shock Transformation.
- Organisms/Systems used: E. Coli, S. Cerevisiae. Mus Musculus, Cell lines.
- Protein extraction and quantification, TCA protein precipitation, Histone Acid Extraction.
- Bacterial and Yeast assays, Yeast genetics.
- Bacterial and Yeast Maximum and Minimum Plates, Yeast Spot assay, silencing assays.
- Skilful at handling radioactive materials.
- Instruments daily used: Spectrophotometer, Typhoon Multimode scanner (phosphor
autoradiography), ChemiDoc MP Imaging System Microscope, Bioruptor Diagenode,
Glomax fluorometer, PCR and Real Time PCR, fume hood, UV Transilluminator, pH meter,
Centrifuge and Ultracentrifuge, Analytical scale, Cross-linker, Incubator, Autoclaves|
|Personal skills keywords: ||Western Blot, Northern Blot, Southern Blot,ChIP, ChIP-seq, RNA-seq|
PCR, semiquantitative sqRT-PCR, Colony PCR, real time qRT-PCR.
Gene and recombinant DNA cloning, recombinant protein for heterologous expression
DNA/RNA extraction, DNA/RNA precipitation, plasmid preparation Mini, Midi and Maxi Prep, skilful in DNA/RNA/Protein handling of different organisms and cell lines.
Agarose and polyacrylamide Gel Electrophoresis, DNA gel purification,Organisms/Systems used: E. Coli, S. Cerevisiae. Mus Musculus, Cell lines.
|Appears in PhD:||SCIENZE PASTEURIANE|
Files in This Item:
|PhD Thesis.pdf||Tesi di Dottorato||20.26 MB||Adobe PDF|
File del Curriculum Vitae:
|CurriculumVitae.pdf|| ||143.48 kB||Adobe PDF|
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